Combined X-linked familial exudative vitreoretinopathy and retinopathy of prematurity phenotype in an infant with mosaic turner syndrome with ring X chromosome

ABSTRACT Background Retinopathy of prematurity (ROP) and familial exudative vitreoretinopathy (FEVR) are two distinct pathologies of retinal angiogenesis with overlapping clinical features. Methods Examination, multimodal imaging, and genetic testing were used to guide diagnosis and treatment. Results We report a combined phenotype of X-linked FEVR and ROP in a 4-month-old girl with mosaic Turner syndrome with ring X chromosome born at 26 weeks gestational age. She was initially diagnosed with atypical ROP with a vitreous band causing a localized traction retinal detachment, inferotemporal to the macula in the right eye, vessels to posterior zone 2 with no clear ridge temporally in the left eye, and fluorescein leakage in both eyes. Due to the suspicion of concurrent FEVR, genetic testing using a vitreoretinopathy panel was performed which revealed a mosaic Turner syndrome associated with 45,X/46,X,r(X), subsequently confirmed by chromosome analysis. The deleted region in the ring X chromosome included the NDP and RS1 genes. The patient was treated with laser photocoagulation of the peripheral avascular retina and sub-Tenon’s triamcinolone injection in both eyes, intravitreal injection of bevacizumab in the left eye, and pars plicata vitrectomy in the right eye. Conclusions In premature neonates with atypical ROP, a clinical suspicion of concurrent FEVR or similar vasculopathy is important and genetic testing may elucidate a genetic etiology, which could influence management and prognosis. Turner syndrome can be connected with co-occurring Mendelian gene disorders, particularly in individuals with mosaicism. The concurrence of FEVR and ROP appears to result in atypical and possibly more severe phenotypes.


Introduction
Retinopathy of prematurity (ROP) and familial exudative vitreoretinopathy (FEVR) are two distinct forms of retinal angiopathy with overlapping clinical features. While the risk factors for ROP include low gestational age, low birth weight and oxygen supplementation, FEVR is an inherited condition resulting from germline pathogenic variants in genes usually in the Wnt pathway required for the growth and development of retinal blood vessels. Recent reports have described a small group of premature infants with atypical ROP with features consistent with FEVR (1)(2)(3). Genetic studies have revealed that many of these infants have germline pathogenic variants in one of the FEVR-associated genes (NDP, FZD4, LRP5, TSPAN12, ZNF408) (4). Premature neonates at risk for ROP, who carry a mutation that causes FEVR, appear to have a more aggressive course of retinopathy than ROP alone (5)(6)(7).
Turner syndrome is the second most common sex chromosomal abnormality in females (following trisomy X), with an estimated incidence of one in two thousand live female births (8). An incomplete or missing X chromosome is characteristic, leading to short stature, congenital cardiac and/or renal defect, ovarian dysgenesis, and neurodevelopmental challenges. The presence and severity of symptoms can vary broadly. Mosaicism occurs in over half of the cases, where there can be two or more cell lines with different genetic make-up; individuals may have a 45,X cell line in addition to 46,XX, 47,XXX, 46, XY, or a cell line involving structural rearrangements such as ring X, isochromosome, or translocation (9,10).
We report a presumed combined phenotype of X-linked FEVR and ROP in a 4-month-old girl with mosaic Turner syndrome with ring X chromosome born prematurely at 26 weeks gestational age.

Case report
A 4-month-old girl initially diagnosed by an outside pediatric ophthalmologist with bilateral retinopathy of prematurity (Stage 3, zone II) and a mild temporal dragging in the right eye was referred to Massachusetts Eye and Ear for evaluation. She was born at a gestational age of 26 weeks and a birth weight of 760 g. The neonatal course included oxygen supplementation for chronic lung disease, delayed milestones, and swelling of the kidneys. Screening for ROP was initially performed at 31 weeks post-conceptual age and showed an immature retina without ROP that progressed to stage 3, zone II ROP at 39 weeks post-conceptual age, per the screening physician. At our institution, an ophthalmic examination at age 4 months (42 weeks post-conceptual age) showed that she could fix and follow with each eye and anterior segment with persistent tunica vasculosa lentis in the right eye.
Examination under anesthesia was performed. Fundoscopy of the right eye revealed retinal vessels in zone 2, vitreous band attached at the optic disc and the retina inferotemporal to the macula with surrounding retinal hemorrhage. In the left eye, retinal vessels were present in posterior zone 2 with no clear ridge and flat retinal hemorrhages in the vascular retina temporally. Wide-field fluorescein angiography (FA) showed retinal vessels ending at zone 2 in both eyes. The area of traction inferotemporal to the macula in the right eye showed leakage and pruning of surrounding vessels. The left eye had peripheral avascular retina and areas of peripheral leakage consistent with retinal neovascularization. B-scan ultrasonography confirmed the localized peripheral traction retinal detachment temporally in the right eye ( Figure 1). The patient was treated with laser photocoagulation of the peripheral avascular retina and sub-Tenon's triamcinolone acetonide injection (Triesence 2 mg in 0.05 mL) around both eyes, and intravitreal injection of bevacizumab (0.625 mg in 0.025 mL) in the left eye.
Since the phenotype was atypical for ROP, genetic testing using a 24-gene panel of inherited vitreoretinopathies was ordered. This is a targeted, next-generation sequencing (NGS) panel with sequencing and copy number analysis of 24 genes associated with inherited vitreoretinopathies (11). These genes included ATOH7, BEST1, CAPN5, COL11A1, COL11A2,  There were no disease-causing sequence variants in the genes analyzed, although a heterozygous variant of uncertain significance in the autosomal recessive gene P3H2 (c.385 G>T, p.(Gly129Trp)) was detected. The targeted NGS gene panel results did, however, detect mosaic possible monosomy X and ring X, which is associated with Turner syndrome. NGS read data suggested approximately 46% of cells had a missing X chromosome, and approximately 54% of cells had 1 to 2 copies of the estimated genomic region of chrX: 46513221-117959147, consistent with mosaicism involving a ring X chromosome with breakpoints at Xp11.23 and Xq24. The estimated genomic region deleted in the presumed ring X encompassed two genes associated with inherited vitreoretinopathies, NDP and RS1. Subsequent chromosome analysis by karyotype on peripheral blood sample detected 45,X in eight out of 20 cells (40%) and a 46,X,r(X) in 12 out of 20 (60%) cells ( Figure 2). Furthermore, chromosomal microarray analysis refined the breakpoints, demonstrating that the mosaic ring X chromosome is composed of material from band p11.23 to band q23, including the X-inactive specific transcript (XIST) gene. The size of the ring X is approximately 69.8 Mb, which we estimate to be a medium-to-large sized ring. Thus, chromosome analysis and microarray testing confirmed the diagnosis of mosaic Turner syndrome.
At age 5 months, the patient underwent pars plicata vitrectomy to cut the tractional vitreous band, sub-Tenon's injection of triamcinolone acetonide (Triesence 20 mg in 0.5 mL) and intravitreal bevacizumab (0.625 mg in 0.025 mL) in the right eye (See supplementary video 1), and additional laser photocoagulation of the remaining peripheral avascular retina posterior to previous laser sessions in the left eye, as the retinal vessel did not progress to the edge of the prior laser as expected (laser #2). One month later, additional laser photocoagulation of the remaining peripheral avascular retina posterior to previous laser sessions in both eyes (laser #2 right eye, laser #3 left) and sub-Tenon's injection of triamcinolone acetonide (Triesence 2 mg in 0.05 mL) were performed around both eyes (injection #3 right eye, injection #2 left eye). There was no residual traction on the retina in the right eye and resolution of the angiographic leakage in both eyes. Further follow-up one month later revealed continued regression of the retinal neovascularization in the right eye and no residual leakage in the left eye (Figure 3).
When evaluated at the Massachusetts General Hospital Turner Syndrome Clinic at age 10 months, the patient was noted to have mild global developmental delay. Her hearing test was normal. Her appearance was typical of Turner syndrome with ptosis, low-set and posteriorly rotated ears, narrow palate, broad chest, and widely spaced nipples. Echocardiography showed normal right ventricular pressures and no evidence of pulmonary hypertension or pulmonary vein stenosis. When re-examined at age 16 months, she had ongoing global developmental delays, truncal hypotonia and extensor hypertonia, consistent with some young individuals with TS with ring X.

Discussion
Our patient highlights the importance of investigating premature infants with atypical or severe ROP for the presence of genetic abnormalities, which in this case, led to dual genetic diagnoses of X-linked FEVR and mosaic Turner syndrome.
John et al. (3) reported an overlap between ROP and FEVR leading to aggressive disease. In our pre-term patient, a FEVR diagnosis was suspected due to certain atypical features not consistent with ROP. These features include the pruning and straightening of the vessels, irregular vascular-avascular junction, asymmetry of disease, focal retinal traction, and neovascularization without a significant ridge (12). In contrast, ROP usually exhibit homogeneity and a regular transition from the vascular to avascular zone demonstrated well by fluorescein angiograms (3).
The clinical suspicion of atypical ROP in this case was the main reason for the workup and for the FEVR diagnosis. Our patient had a deletion including the entire NDP gene on the ring X chromosome. Pathogenic variants of the NDP gene are known to cause X-linked FEVR phenotype in males (13,14). Many genetic abnormalities on the X chromosome are well tolerated in females because of the preferential inactivation of the abnormal X which may balance the genetic material. In cases of ring X chromosome, the phenotype can be more severe compared to those with non-mosaic Turner syndrome (45,X). In our case, the ring X chromosome paradoxically included the XIST gene responsible for the initiation of X inactivation. Severe phenotype can still occur in the context of ring X chromosome with XIST expression due to the partial expression of the XIST gene leading to incomplete inactivation of r(X) or the inactivation of the normal X chromosome during embryogenesis (15). This latter phenomenon is called skewing of X inactivation and results in phenotypic expression of X-linked recessive diseases in females (16). It remains unclear why individuals with ring X have an increased chance of having more significant developmental delays and possibly intellectual disability compared to those with TS without ring X. Although exceptions exist in the literature and in our clinical experience, this patient has typical developmental delays, and longitudinal follow-up is needed to determine if there will be intellectual disability.
Interestingly, female carriers of NDP pathogenic variants can have a spectrum of retinal features including mild clinical signs of FEVR (17)(18)(19)(20), most likely due to the inactivation of the normal X chromosome in a subset of cells. Due to the normal copy of the normal copy of the NDP gene on the intact X chromosome, the most likely explanation of the observed phenotype in this patient is due to skewing of X inactivation, but this cannot be definitively proven.
The developmental outcome cannot be predicted solely by the presence or absence of XIST. The level of mosaicism, the ring size, and gene content are additional factors that influence the severity of the phenotype, but there is variable karyotypephenotype correlation. Furthermore, the impact of mosaicism on clinical manifestation cannot be demonstrated accurately because mosaicism is variable between tissues. The level of mosaicism detected in the peripheral blood may not be an accurate representation of that in the retina. Thus, disease expression may vary remarkably between tissues, and eyes can exhibit asymmetric findings depending on retinal cell karyotypes.
Based on the genetic findings of X chromosome mosaic monosomy 45,X/46,X,r(X) (p11.23q24) and her developmental delay, our patient was diagnosed with mosaic Turner syndrome (10). Jones et al. showed that 1% to 4% of patients with Turner syndrome present with a co-occurring condition, including X-linked disorders, with a higher prevalence in patients with mosaicism of the X chromosome compared with 45, X (80% vs. 50%) (21). Among these dual diagnoses, ocular pathologies have been reported (22,23). Recently, Laura et al. reported a case of FEVR in the setting of Turner syndrome with a LRP5 gene mutation (24). There have been no prior reports of NDP pathogenic variant and Turner syndrome.
Interestingly, this patient was found to have a deletion of the entire RS1 gene, which is associated with X-linked retinoschisis (×LRS) (25). However, examination showed no macular or peripheral retinoschisis. Although the clinical significance of this finding is unclear, possible explanations may be the presence of a normal allele of RS1 gene on the remaining X chromosome and that the karyotypes of retinal cells retain the RS1 gene in the context of mosaic Turner syndrome. It is noteworthy that Rajendran et al. suggested a functional relationship between NDP and RS1 genes through FZD4 protein interactome, including the perilipin 2 (PLIN2) and the mitogen-activated protein (MAP) kinase signaling pathways (26). Additional studies need to be performed to determine if this is a significant interaction clinically. Moreover, there was absence of any phenotype related to the contiguous gene deletion syndrome associated with NDP that includes MAOA/B and EHFC2, which are presumably also encompassed by this deletion (27). The phenotypes are only present in the tissues that express the ring X and silence the normal X. Depending on the tissues needs, the cells that express the ring X may not be viable. Hence, only the cells expressing the normal X would make up those tissues leading to a normal phenotype in those tissues. In addition, the ring X may not be stable during meiosis, which may be partially responsible for some variation in phenotypes.
Treatment for our patient was similar to the typical management of ROP and FEVR with laser to the avascular retina and intravitreal bevacizumab injection to regress the neovascularization with the use of sub-Tenon's triamcinolone to decrease inflammation associated with the laser. Bevacizumab injection was not initially performed in the right eye due to the risk of rapid regression and contraction (or crunch) of the area of retinal neovascularization that may lead to an increase in the vitreous traction and traction retinal detachment. The surgical procedure in the right eye consisted of a vitrectomy to sever and trim the tractional vitreous bands. The hyaloid was not lifted to avoid retinal breaks. Subsequent FA and B-scan ultrasound demonstrated resolution of the retinal neovascularization and traction retinal detachment.
In summary, this case represents the first known report of presumed combined phenotype from X-linked FEVR and ROP, with the X-linked FEVR thought to be expressed in this girl by the mosaic Turner syndrome with ring X chromosome. This highlights the importance of maintaining a clinical suspicion of FEVR in premature neonates with atypical retinopathy.